Polycaprolactone nanofibrous mesh reduces foreign body reaction

International Journal of Nanomedicine

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Polycaprolactone nanofibrous mesh reduces foreign body reaction and induces adipose flap expansion in tissue engineering chamber This article was published in the following Dove Press journal: International Journal of Nanomedicine 5 December 2016 Number of times this article has been viewed

Lin Luo 1,* Yunfan He 1,2,* Qiang Chang 1,2 Gan Xie 1 Weiqing Zhan 1 Xuecen Wang 1 Tao Zhou 1 Malcolm Xing 2,3 Feng Lu 1 Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China; 2Department of Mechanical Engineering, Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada, 3Children’s Hospital Research Institute of Manitoba, Winnipeg, Manitoba, Canada 1

*These authors contributed equally to this work

Abstract: Tissue engineering chamber technique can be used to generate engineered adipose tissue, showing the potential for the reconstruction of soft tissue defects. However, the consequent foreign body reaction induced by the exogenous chamber implantation causes thick capsule formation on the surface of the adipose flap following capsule contracture, which may limit the internal tissue expansion. The nanotopographical property and architecture of nanofibrous scaffold may serve as a promising method for minimizing the foreign body reaction. Accordingly, electrospinning porous polycaprolactone (PCL) nanofibrous mesh, a biocompatible synthetic polymer, was attached to the internal surface of the chamber for the reducing local foreign body reaction. Adipose flap volume, level of inflammation, collagen quantification, capsule thickness, and adipose tissue-specific gene expression in chamber after implantation were evaluated at different time points. The in vivo study revealed that the engineered adipose flaps in the PCL group had a structure similar to that in the controls and normal adipose tissue structure but with a larger flap volume. Interleukin (IL)-1β, IL-6, and transforming growth factor-β expression decreased significantly in the PCL group compared with the control. Moreover, the control group had much more collagen deposition and thicker capsule than that observed in the PCL group. These results indicate that the unique nanotopographical effect of electrospinning PCL nanofiber can reduce foreign body reaction in a tissue engineering chamber, which maybe a promising new method for generating a larger volume of mature, vascularized, and stable adipose tissue. Keywords: polycaprolactone nanofibrous mesh, topography, porous structure, adipose tissue regeneration, foreign body reaction

Introduction Correspondence: Feng Lu Department of Plastic and Aesthetic Surgery, Nanfang Hospital of Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, Guangdong 510515, People’s Republic of China Email [email protected] Malcolm Xing Department of Mechanical Engineering, Biochemistry and Medical Genetics, University of Manitoba and Children’s Hospital Research Institute of Manitoba, Winnipeg, MB, Canada Email [email protected]

The reconstruction of soft tissue defects following trauma (eg, severe burns), tumor resection (eg, mastectomy), or aging still presents a major challenge in plastic and reconstructive surgery.1,2 Current techniques used to reconstruct large soft tissue defects include artificial implants or autologous tissue transplantation, which are associated with certain limitations, including resorption and donor site morbidity.3–5 Tissue engineering offers an alternative to these suboptimal techniques, but vascularization is a major current limitation to the size, maintenance, and quality of engineered tissue. Based on Fick’s diffusion theory, cells at a distance of more than 200 μm from a blood vessel or capillary in vivo are either inactive or necrotic due to the limitations of nutrient diffusion.6–8 In 2003, the tissue engineering chamber technique was introduced, which involves embedding a vascularized pedicled adipose flap in a chamber, making it possible to generate mature, vascularized, and 6471

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http://dx.doi.org/10.2147/IJN.S114295

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Luo et al

transferable adipose tissue.9 In 2011, the initial 5 mL of adipose flap successfully expanded to 56.5 mL using large volume chamber (78.5 mL) in pig model, providing clinically relevant volumes of tissue for soft tissue construction.10 Despite the advancement of tissue engineering chamber technique in recent years,11–13 the final maximum volume of the flap was still unable to fill the chamber thoroughly for unknown reasons. Efforts have been made to induce larger volume of the flap, including the usage of a larger volume chamber, exogenous growth factors, and extracellular matrix scaffolds.14–17 However, these methods are associated with the generation of toxic degradation products, risk of tumor formation, and high proportion of fibrous tissue in chambers. Thus, more feasible approaches are still needed to induce a larger volume adipose flap in tissue engineering chamber. Various implant materials are used for the reconstruction of soft tissue defects as well as for aesthetic breast augmentation, with silicone being one of the most popular and accepted implantable biomaterials. Like all nonabsorbable implants, silicone may cause fibrous capsule formation of varying thickness.18,19 Myofibroblasts are contractile fibroblasts found in fibrous capsule, which provide a contractile force and decrease the surface area of the capsule, leading to the capsule contracture over time.20 Moreover, clinical study of capsular contractures after aesthetic breast augmentation revealed that the average tensile strength of the capsule was 44±38 N,21 and the intracapsular pressure correlated positively with the degree of capsule thickness.22 Chambers used in the tissue engineering chamber technique are mainly made of silicone, and the formation of the thick fibrous capsule on the adipose flap surface in silicone chambers has been frequently reported.10,11,13,23 Therefore, we speculated that the foreign body reaction induced by implanted silicone chambers would lead to capsule formation on the adipose flap surface and subsequent capsule contraction, which is similar to what happened with human silicone implants; this could be one of the major factors contributing to the limited final maximum volume of an engineered flap. Reducing the extent of foreign body reaction in silicone chamber would induce decreased fibrous capsule formation and reduce the degree of contraction on the adipose flap surface, which would ultimately lead to a larger volume of adipose flap. Reducing the extent of foreign body reaction induced by subcutaneous implants remains a hot topic, with topographical modification being a research focus for minimizing the reaction in the past few years. Recent studies have indicated that material topographical features have a dramatic effect on foreign body reaction,24,25 and a porous structure tends to result in a moderate tissue response.26,27 Polycaprolactone 6472


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(PCL) nanofibrous scaffold, a biocompatible synthetic polymer with a relatively long degradable period, was reported to have a porous structure and was proven to be effective in suppressing foreign body reaction and fibrous capsule formation in vivo.28,29 With the decrease in intracapsular pressure, the neoformed tissue induced by a chamber undergoes further expansion. To test our hypothesis, we fabricated porous PCL nanofibrous mesh using the electrospinning technique and attached it to the internal surface of a silicone chamber before implantation. Inflammation level, cell proliferation, collagen quantification, capsule thickness, and adipose tissue-specific gene expression in a chamber after implantation were evaluated at different time points.

Materials and methods PCL nanofibrous mesh preparation and tensile test PCL (Mw:80,000 g/mol, Sigma-Aldrich, St Louis, MO, USA, 10 wt %) solution was prepared in dimethylformamide/ dichloromethane (Sigma-Aldrich 1:4, v/v) solvent mixture by gentle magnetic stirring at room temperature until completely dissolved.29,30 The solution was loaded into 10 mL Luer-lock syringe with 25-gauge needle, and the infusion rate was regulated by a syringe pump (Harvard PHD syringe pump, Harvard Apparatus, Holliston, MA, USA). The infusion rate was set to 1 mL/h; the distance between the needle tip and steel mash collector was 18 cm.31 High direct current voltage (Gamma High Voltage Research, Ormond Beach, FL, USA) was applied to the PCL solution. The electrospinning process was conducted at room temperature (25°C±1°C) and with the humidity maintained at 45%. After electrospinning, the PCL nanofibrous mesh was torn off, and 1 mm thickness sheets were used at following in vivo test. Mechanical test of PCL nanofibrous mesh was performed using Instron tensile tester (5965; Illinois Tool Works Inc., Glenview, IL, USA) at ambient environment. The sample was cut with scissors into 15×10 mm2 rectangular shape and the thickness was measured with a vernier caliper, then the sample was placed between two crossheads, resulting in specimens dimension of 10×10×0.1 mm3, and the crosshead speed was set to 10 mm/min. Data, including stress (in MPa) and elongation (%), were automatically calculated by the Instron’s Bluehill 2 software (Instron, High Wycombe, UK).

Scanning electron microscopy evaluation of PCL nanofibrous mesh structure PCL nanofibrous mesh was cut into square samples (1×1 cm2). The surface characteristics of the scaffolds were International Journal of Nanomedicine 2016:11

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Tissue engineering chamber generates engineered adipose tissue

further investigated using the scanning electron microscope (Hitachi S-3000N; Hitachi Ltd., Tokyo, Japan). The mesh were mounted on carbon tape and coated with gold prior to imaging. An acceleration voltage of 20 kV was used and images were observed using Canon digital camera (Canon Inc., Tokyo, Japan).

Animals and tissue engineering chambers Adult male Sprague Dawley rats weighing 350–400 g were used for this study. All rats were housed on a 12-hour light/ dark schedule, and received a stock diet and water ad libitum. All experiments were performed under National Institutes of Health protocols and were approved by the Ethics Committee of Southern Medical University. All operations were performed with general anesthesia (nembutal administered intraperitoneally at 60 mg/kg body weight). Cylindrical silicone chambers were manufactured by the clinical laboratory of Nanfang Hospital (Guangzhou, People’s Republic of China). The perforated chambers were 15 mm long, with an internal diameter of 12 mm and a volume of 1.7 mL.

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Experimental design and surgical techniques Sprague Dawley rats (n=66) were subdivided into experimental (n=33) and control groups (n=33). In the PCL group, the PCL nanofibrous mesh was attached to the internal surface of the chamber with 6-0 sutures (Figure 1A, right). Chambers without PCL nanofibrous mesh served as control (Figure 1A, left). A pedicled adipose flap based on the superficial inferior epigastric vessels of standardized volume was dissected in situ (Figure 1C). Chambers were inserted into both groins of each rat (Figure 2A and B) as described in Dolderer et al’s study.23 Briefly, the groins of rats were depilated with a hair remover, and the skin was then decontaminated with chlorhexidine and alcohol. A longitudinal incision was made in the groin to reveal the bilateral groin adipose pad. The pedicled adipose flap was embedded into the silicone chamber with a proper position to avoid the vasospasm, and then the chamber was anchored to underlying muscle with a 7-0 nylon microsuture. The wounds were closed with 4-0 sutures.

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7HQVLOHVWUDLQPPPP Figure 1 The rat chamber model and microstructure, stress–strain curve of PCL nanofibrous mesh. Notes: (A) Cylindrical silicone chambers (left), PCL nanofibrous mesh was attached to the internal surface of the chamber (right). (B) SEM images of PCL nanofibrous mesh demonstrated the pore structure and nano-sized fibers. (C) A vascularized pedicled adipose flap based on the superficial inferior epigastric vessels of standardized dimensions was dissected in situ. (D) The stress–strain curve of PCL nanofibrous mesh. Abbreviations: PCL, polycaprolactone; SEM, scanning electron microscopy.

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Figure 2 Animal model and harvested adipose flap. Notes: (A) PCL nanofibrous mesh was attached to the internal surface of the cylindrical silicone chamber in the PCL group. Chambers without PCL nanofibrous mesh served as control. (B) Chambers embedded with a pedicled adipose flap were inserted into both groins of rats. (C) Adipose flap harvested at chamber removal at different time points. Abbreviation: PCL, polycaprolactone.

Tissue harvesting Rats were anesthetized at weeks 1, 2, 4, 8, and 12. Adipose flaps were harvested from chambers (Figure 2C), and their volumes were measured based on the volume of fluid displacement in normal saline. Each harvested sample was prepared for the following analyses. The anesthetized animals were killed by overdosed sodium pentobarbital injection (5 mL of a 20 mg/mL solution).

Histological examination The tissue samples were fixed in 4% paraformaldehyde, dehydrated, and paraffin embedded for routine histological processing. Tissue blocks were serially sectioned (4 µm sections) along the longitudinal axis and stained using routine hematoxylin and eosin protocol, after which they were assessed under microscope and photographed with the digital camera (Olympus Corp., Tokyo, Japan).

Whole-mount staining Harvested adipose samples were cut into 0.5–1 mm pieces after sacrifice and incubated with the following reagents for 30 minutes: BODIPY 558/565 (dilution 1:300; Molecular Probes, Eugene, OR, USA) to stain adipocytes, Alexa Fluor 488-conjugated isolectin GS-IB4 (dilution 1:200; Thermo Fisher Scientific) to stain endothelial cells, and Hoechst 33342 (dilution 1:500; Thermo Fisher Scientific) to stain

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nuclei. The samples were then washed and directly observed with a confocal microscope (FluoView™ FV1000, Olympus Corp., Tokyo, Japan).

Immunohistochemistry for Ki67 Sections were immunolabeled at different time points (weeks 4, 8, 12) from the samples of both groups. The rabbit anti-rat Ki67 antibody (Abcam, Cambridge, MA, USA) was used to stain cells for proliferation. Briefly, paraffin sections were dewaxed and hydrated before immunohistochemical staining. Sections were washed in Tris-buffered saline and blocked with rabbit serum. Then, the sections were incubated with a primary antibody at 4°C overnight. After three wash steps, sections were incubated with the biotinylated secondary antibody (goat anti-rabbit IgG; Santa Cruz Biotechnology Inc., Dallas, CA, USA), diluted 1:200 in phosphate-buffered saline for 1 hour at room temperature. Slides were scored by two independent observers and photographed by Olympus DP71 digital camera (Tokyo, Japan).

Collagen quantification For determination of collagen deposition in samples, adipose tissue obtained at all five time points from two groups were stained with Masson’s trichrome. Briefly, sections were deparaffinized in xylene, rehydrated in graded ethanol, and postfixed in Bouin’s fixative for 1 hour at 55°C. The nuclei and


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collagen were sequentially stained with equal volumes of ferric chloride solution and alcoholic hematoxylin and trichrome solution, respectively. The total collagen content was reported as a percentage of the aniline blue staining divided by the total tissue area of the section using ImageJ software (National Institutes of Health, Bethesda, MD, America).

Quantitative real-time polymerase chain reaction For real-time polymerase chain reaction (PCR) analysis, sample RNA was isolated from the chamber contents using Trizol and SYBR Green quantitative PCR SuperMix (Thermo Fisher Scientific), followed by reverse transcription. The complementary DNA samples were measured using customized TaqMan Array Plates (Thermo Fisher Scientific). Quantitative real-time PCR was carried out according to the TaqMan method (50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minutes) with previously designed primers (Thermo Fisher Scientific) and was used for expression analysis. Relative expressions were calculated by cycle threshold method using β-actin as an endogenous reference gene. The mouse-specific primers used to amplify messenger RNA sequences were: Transforming growth factor beta 1 (TGF-β1) forward 5′-TGCTTCAGCTCCACAGAGAA-3′; and reverse: 5′-TGGTTGTAGAGGGCAAGGAC-3′; Peroxisome proliferator-activated receptor gamma (PPARγ) forward 5′-CTGGCCTCCCTGATGAATAA-3′; and reverse: 5′-GGCGGTCTCCACTGAGAATA-3′; IL-1β forward 5′-AGGCAGTGTCACTCATTGTGG-3′; and reverse: 5′-TAGCAGGTCGTCATCATCCC-3′; IL-6 forward 5′-GCCTTCTTGGGACTGATGTT-3′; and reverse: 5′-ACTGGTCTGTTGTGGGTGGT-3′; β-actin forward 5′-ACCCGCGAGTACAACCTTCTT-3′; and reverse: 5′-TATCGTCATCCATGGCGAACT-3′.

Statistical analysis

Data were expressed as mean ± SD. results were analyzed with two-way analyses of variance (SPSS, version 21; IBM Corporation, Armonk, NY, USA). Furthermore, an independent Student’s t-test of two groups at one time point was performed. A value of P,0.05 was considered statistically significant.

Results Analysis of PCL nanofibrous mesh pore size, structure, and mechanical property Scanning electron microscope was used to evaluate the size, structure, and diameters of the fibers within the film.


Tissue engineering chamber generates engineered adipose tissue

The PCL nanofibrous mesh exhibited a porous structure, and the diameter of the fibers was 550±69 nm. The mean porosity was 89.4%±2% (Figure 1B). The stress–strain curve was presented in Figure 1D. The mean value of Young’s modulus was 2.4±0.4 MPa, and the average of elongation was 190%±15%. These values are consistent with previous reports of PCL nanofibrous mesh.32,33

Morphometry and volume analysis at chamber removal Two animals died owing to infection. No significant inflammation or tissue necrosis was found at the silicone chamber implantation site. The PCL nanofibrous mesh was preserved perfectly and was attached to the internal surface of the silicone chamber in all animals in the PCL group. At week 4, after chamber removal, the PCL group consisted largely of a fragile, white, gel-like fibrin exudation surrounding the flap (Figure 3A) with small adipocytes detectable in the connective tissue (Figure 3B and C). However, by week 8, new adipose tissue that had formed with newly formed blood vessels could be detected on the surface of fibrin exudation (Figure 3D and E). Similar features were observed in the constructs of the control group at 4 and 8 weeks, except that these constructs had a tougher tissue texture. By week 12, both groups developed vascularized and well-organized adipose tissue, with almost nonexistent fibrin exudation at the outermost layer (Figure 3G, H, and I). Quantification of the flap volume revealed a gradual volume increase in both groups from week 0 to week 2. The volume of the adipose flap increased from a baseline value of 0.100±0.060 mL to 0.140±0.012 mL in the PCL group and from 0.093±0.007 mL to 0.147±0.009 mL in the control group. The flap then showed a sharp increase to 0.357±0.029 mL and 0.250±0.023 mL at week 4, and further increased to 0.440±0.026 mL and 0.330±0.023 mL at week 8 in the PCL and control groups, respectively. The flap volume of the PCL group was significantly larger than the control group as of week 4 (P,0.05). The constructs volume in both groups appeared to reach full development by week 8 (Figure 4).

Visualization of the tissue architecture in engineered adipose tissues Whole-mount staining was applied to visualize the tissue architecture and demonstrate the adipocyte and blood vessel interactions in the engineered tissue formed in the PCL group at week 12. For comparison, the control group was treated in the same manner.


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Figure 3 Engineered adipose flap in the PCL group chamber and hematoxylin and eosin-stained section at different time points. Notes: The tissue of PCL group from week 4 consisted largely of a fragile, white, gel-like fibrin exudation surrounding the flap (A) with small adipocytes (inset) detectable in the connective tissue (B). By 8 weeks, new adipose tissue had formed (E) and small blood vessels could be detected on the surface of the fibrin exudation (D). Similar observations were made ain 4- and 8-week constructs of control groups (C, F), By week 12, both groups had developed vascularized and well-organized adipose tissue, with almost nonexistent fibrin exudation at the outermost layer (G, H, I). Scale bar =100 μm. Abbreviation: PCL, polycaprolactone.

At baseline, capillaries were observed between mature adipocytes in both groups. More mature and integrated structure of adipose tissue could be observed in the PCL group than the control group at week 12 (Figure 5).

most of the Ki67+ cells located around vessels. However, the number of Ki67+ cells decreased sharply at week 8, and almost no Ki67+ cells were found at week 12 (Figure 6).

Angiogenesis and cell proliferation

Collagen deposition and changes in capsule thickness

Proliferation marker Ki67 staining showed similar dynamic changes in Ki67+ cells in both groups. At week 4, abundant Ki67+ cells were scattered throughout the tissues, with

To demonstrate the collagen deposition in the tissue, adipose tissue harvested from both groups was stained with Masson’s trichrome, and the areas of collagen staining (blue) were 3&/JURXS

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Figure 4 Volume changes in flaps at different time points. Notes: There was a sharp volume increase (both groups) from week 2 to week 4. The constructs volume in both groups appeared to have reached their full development by 8 weeks. Statistical analysis demonstrated that the flap volume of the PCL groups were significantly larger than the control group since week 4 (*P,0.05). Abbreviation: PCL, polycaprolactone.

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